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Related Experiment Videos

Site-directed mutagenesis alters DnaK-dependent folding process.

Kazuaki Yoshimune1, Nobuyoshi Esaki, Mitsuaki Moriguchi

  • 1Department of Applied Chemistry, Faculty of Engineering, Oita University, Dannoharu 700, Oita 870-1192, Japan.

Biochemical and Biophysical Research Communications
|November 30, 2004
PubMed
Summary

Overproducing Alcaligenes d-aminoacylase (A6-d-ANase) caused aggregation, but coexpressing DnaKJE or mutating R354 to lysine improved soluble expression. This reveals DnaK-dependent folding is altered by mutagenesis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Overproduction of Alcaligenes d-aminoacylase (A6-d-ANase) in Alcaligenes xylosoxydans subsp. xylosoxydans A-6 leads to protein aggregation.
  • Coexpression of the DnaK-DnaJ-GrpE (DnaKJE) chaperone system facilitates soluble expression of overproduced proteins.

Purpose of the Study:

  • To investigate the role of the DnaKJE chaperone system in the soluble expression of A6-d-ANase.
  • To determine if site-directed mutagenesis of A6-d-ANase can improve its solubility and activity.

Main Methods:

  • Expressing the A6-d-ANase gene in Escherichia coli dnaK mutant dnaK756.
  • Coexpressing DnaKJE with A6-d-ANase in the mutant strain.
  • Performing site-directed mutagenesis on the R354 residue of A6-d-ANase.

Related Experiment Videos

  • Assessing enzyme activity in the soluble protein fraction.

    • Main Results:

    • Expression of A6-d-ANase in the dnaK mutant resulted in low soluble activity.
    • Coexpression of DnaKJE or substitution of R354 with lysine restored soluble activity.
    • The guanidino group of R354 appears to hinder proper folding without DnaK, a disturbance resolved by DnaK binding.

    Conclusions:

    • The DnaK-dependent folding process of A6-d-ANase is influenced by the R354 residue.
    • Site-directed mutagenesis at R354 can overcome folding issues in the absence of DnaK.
    • This study provides the first evidence of altered DnaK-dependent enzyme folding through site-directed mutagenesis.